Deciphering pachytene piRNA function PROJECT SUMMARY / ABSTRACT An enigmatic class of small RNAs appears at the pachynema of Meiosis I of mammalian spermatocytes. They are processed from long, non-coding RNAs, bind to Piwil1 (in mouse commonly known as Miwi) protein, and are termed pachytene piwi-interacting (pi) RNAs. Miwi/piRNAs are essential for spermiogenesis and male fertility. Our laboratory discovered that in diverse species, Piwi proteins loaded with piRNAs are symmetrically dimethylated on specific arginines by the methylosome, and mediate interaction with Tudor domain containing (Tdrd) proteins. Miwi binds directly to Tdrd6, a protein that contains six canonical Tudor domains, and together form the core of the chromatoid body, a large, cytoplasmic, non-membrane bound structure that contains numerous mRNAs along with pachytene piRNAs. Pachytene piRNAs are very abundant; they are not conserved even among closely related species; their sequence diversity is enormous; and their function still remains a mystery. Hypotheses about their roles need to reconcile two seemingly contradictory properties: like microRNAs, pachytene piRNAs are loaded to an Argonaute protein, Miwi, and can serve as guides to bind RNA targets. Unlike microRNAs, their sequence diversity is so enormous that they can bind any mRNA at multiple sites, thus losing sequence-driven specificity. In this application we propose a radically new conceptual framework to crack the enigma of pachytene piRNA function. We will test the hypothesis that pachytene piRNAs play a critical role in sorting transcripts, by dynamically trapping non-spermiogenic mRNAs in Miwi-piRNA-Tdrd6 assemblies, which form the core of the chromatoid body. In our model, multivalent interactions between Miwi/piRNAs, which bind with partial complementarity to mRNAs, and between Miwi and the multiple Tudor domains of Tdrd6, nucleate the chromatoid body that sequesters trapped mRNAs for eventual elimination during spermiation. The model predicts that longer mRNAs are preferentially trapped as they contain more binding sites for piRNAs, while spermiogenic mRNAs that need to be translated to drive spermatid differentiation should be shorter to avoid trapping. We will also test whether the multivalent interactions in Tdrd6-Miwi/piRNA-mRNA assemblies lead to liquid-liquid phase separations that underlie the formation of the chromatoid body. We are confident that the multiple, orthogonal, in vitro and in vivo approaches that we propose will illuminate expected and unexpected outcomes and truly uncover the elusive function of mammalian pachytene piRNAs.